Abstract

The effect and underlying mechanism of vitamin A on norovirus infection are largely unknown. This study aimed to investigate how vitamin A administration affects the gut microbiome after norovirus infection. Here, we demonstrate that treatment with either retinol or retinoic acid (RA) inhibits murine norovirus (MNV) replication using both in vitro and in vivo models. Compositional changes in the gut microbiome associated with RA administration and/or norovirus infection were also investigated. Oral administration of RA and/or MNV significantly altered intestinal microbiome profiles. Particularly, bacterial species belonging to the Lactobacillaceae families were remarkably increased by MNV inoculation and RA administration, suggesting that the antiviral effects of RA occur via the modulation of specific microbiota. The antiviral causal effect of Lactobacillus was identified and demonstrated using in vitro models in RAW264.7 cells. The antiviral immune response to MNV was mediated by IFN-β upregulation. This study represents the first comprehensive profiling of gut microbiota in response to RA treatment against norovirus infection. Moreover, we conclude that the abundance of Lactobacillus through gut microbiota modulation by RA is at least partially responsible for norovirus inhibition.

Treatment with vitamin A effectively inhibited norovirus replication in vitro.

(a) Inhibition of MNV replication by retinol treatment. MNV (MOI 0.01) was infected into RAW 264.7 cells. Cells were then treated with retinol, and MNV replication was measured using a plaque assay. (b) Expression of the human norovirus genome. HG23 cells were incubated with various concentrations of retinol for 24–72 h. Total RNA of the human norovirus genome was quantified by real-time RT-PCR. In vitro tests were repeated three times. Significance was analyzed by the Mann–Whitney U-test and compared to the negative control. *p < 0.05. Retinol did not exhibit cytotoxicity under the conditions used in this experiment.

(a) Beta diversity in groups categorized by RA administration and MNV inoculation was assessed by weighted and unweighted principle coordinate analysis (PCoA). Five groups categorized by RA administration and MNV inoculation were clearly clustered in the weighted, but not the unweighted, analysis. This result indicated that the abundances of certain bacterial taxa were changed by MNV inoculation following RA administration. (b) PCoA of KEGG pathways predicted by PICRUSt. MNV inoculation and RA administration also affected the KEGG pathway categories, as well as the bacterial diversity.

Significant bacterial abundances according to RA administration and MNV inoculation.

(a) Characterization of bacterial abundance by MNV and RA. Significant differences were identified by LEfSe analysis as a p value <0.05 in both the Kruskal–Wallis test (among classes) and Wilcoxon test (between subclasses). The threshold logarithmic LDA score was 3.0. NC: negative control. RA was suspended in corn oil, and administered orally. (b) Abundance of Lactobacillus by RA administration and MNV infection. Among abundant bacteria from the LEfSe analysis, the abundance of Lactobacillus is shown separately. Bacterial abundances were decreased by MNV infection and increased by RA administration after MNV inoculation.

Inhibition of MNV replication by Lactobacillus strains in RAW264.7 cells.

MNV replication was significantly inhibited by four Lactobacillus strains and retinol (50 U/mL). Lactobacillus (estimated at 1 × 105 CFU by OD) was inoculated into MNV-inoculated RAW 264.7 cells (0.01 MOI) for 24 h in the absence of retinol. All Lactobacillus strains significantly decreased the replication of MNV. LPS (10 ng/mL), used as a control, showed an antiviral effect similar to that of Lactobacillus. After freezing and thawing twice, a plaque assay was performed to quantify MNV. Different superscript letters indicate significant differences (p < 0.05) according to Duncan’s post hoc test.